The present invention relates to a method and apparatus for measuring the length of a polynucleotide or biomolecular such as DNA, and is particularly concerned with measuring the length of a not less than 50, especially 100 kilobase long polynucleotide.
Such polynucleotide length measurement is one of the gene analysis means useful in fields like medical chemistry, biochemistry and genetics.
In the past, measuring the base length (or molecular weight) of a long DNA chain has been based on the use of gel electrophoresis migration which provides electric fields in a certain direction. Polyacrylamide gel is effective in separating DNA several bases to 2 kilobases long, and agarose gel is in use for separating DNA 1 to 20 kilobase long.
In conventional electrophoresis using electric fields in a certain direction, molecular sizes are separated in polyacrylamide gel or agarose gel according to the spatial spread of the molecules. To be more specific, the separation is based on whether spatially spread molecules pass through gel meshes. Larger molecules do not pass but become elongated. After all, these elongated molecules are separated according to their shorter diameters. In this case, however, the difference in the size of these elongated DNA molecules is no more than the difference in their seeming lengths. This is why long DNA cannot be separated with molecular sieves. Another conventional technique, pulsed field electrophoresis, varies pulsatively the electric field direction for electrophoresis in agarose gel in order to separate molecules tens of megabases to several megabases long.
A more detailed description of measuring by separation not less than 100 kilobase long DNA may be found in PCT International Publication WO 84/02001 and Nucleic Acid Research, vol. 16, pp. 7563-7582, by B.W. Birren, 1988. In this method, a mixture of giant DNA fragments subject to measurement is injected into wells in an electrolyte-containing gel matrix made of network polymers like agarose, the DNA is allowed to migrate as the direction of electric fields is varied pulsatively, and the DNA is separated according to the size. For instance, when migration is performed for 95 hours in a 0.7% concentration agarose matrix, the direction of 2 V/cm electric fields being varied an angle of +106 degrees with the migration direction every 30 minutes, it is possible to separate 3 to 10 megabase long DNA. The length of the DNA is considered to be measurable on the basis of the bandwidth of the separated DNA with about 10% errors. This pulsed field electrophoresis employs the dependence on elongated molecule lengths of the time taken by varying the direction of the orientation of the molecules by changing the direction of the electric fields (see pp. 364-370, Jikken Igaku, vol. 5 by Hasegawa and Kikuchi (1987)).
The above conventional technique makes it possible to obtain information on the length of very large DNA with pulsed field separation. But the problem is that the longer DNA, the longer time its separation takes. For example, it generally takes 3 or 4 days to separate 3 to 10 megabase long DNA. This conventional technique separates DNA according to the size, namely, the length difference while DNA termini travel through the gel, popping out from and popping in between network molecules composing the gel matrix. Naturally, as the distance of the travel through the gel is shorter, the separation worsens. Moreover, when migration is provided by high electric field intensity, heating impairs separation. Thus it is unfeasible that the conventional technique shortens the analysis time. Since the longer DNA, the longer migration it requires, the technique is not suitable for the practical use for separating not less than tens of megabases long DNA. Besides, the fact that the longer DNA, the longer migration it needs, and the larger bandwidth the separated DNA has deteriorates the precision of the length separation.
Furthermore, the above conventional electrophoresis including pulsed field electrophoresis separates and measures the measurement subject as a group of different molecule sizes, using the difference between molecule sizes and between electrophoresis rates. This deteriorates separation as the molecules are longer, and increases the volume that groups of the same molecule size occupy for a cause such as diffusion, resulting in inefficient separation of long molecules like DNA.
Pulsed field electrophoresis, which varies the electric field direction, uses radio isotope labels or ethidium bromide staining for the purpose of detection. To ensure sufficient detection sensitivity, many molecules (copies) of the same length are necessary and need to be prepared. The resolution for DNA size provided by this technique is not very high because of influences such as molecular heat diffusion, and the molecule length measurement precision is low.